Limits...
Structure and dynamics of the membrane attaching nitric oxide transporter nitrophorin 7.

Knipp M, Ogata H, Soavi G, Cerullo G, Allegri A, Abbruzzetti S, Bruno S, Viappiani C, Bidon-Chanal A, Luque FJ - F1000Res (2015)

Bottom Line: However, a chain-like arrangement in the crystal lattice due to a number of head-to-tail electrostatic stabilizing interactions is found in NP7.Fast and ultrafast laser triggered ligand rebinding experiments demonstrate the pH-dependent ligand migration within the cavities and the exit route.Finally, the topological distribution of pockets located around the heme as well as from inner cavities present at the rear of the protein provides a distinctive feature in NP7, so that while a loop gated exit mechanism to the solvent has been proposed for most nitrophorins, a more complex mechanism that involves several interconnected gas hosting cavities is proposed for NP7.

View Article: PubMed Central - PubMed

Affiliation: Max-Planck-Institut für Chemische Energiekonversion, Mülheim an der Ruhr, 45470, Germany.

ABSTRACT
Nitrophorins represent a unique class of heme proteins that are able to perform the delicate transportation and release of the free-radical gaseous messenger nitric oxide (NO) in a pH-triggered manner. Besides its ability to bind to phospholipid membranes, the N-terminus contains an additional Leu-Pro-Gly stretch, which is a unique sequence trait, and the heme cavity is significantly altered with respect to other nitrophorins. These distinctive features encouraged us to solve the X-ray crystallographic structures of NP7 at low and high pH and bound with different heme ligands (nitric oxide, histamine, imidazole). The overall fold of the lipocalin motif is well preserved in the different X-ray structures and resembles the fold of other nitrophorins. However, a chain-like arrangement in the crystal lattice due to a number of head-to-tail electrostatic stabilizing interactions is found in NP7. Furthermore, the X-ray structures also reveal ligand-dependent changes in the orientation of the heme, as well as in specific interactions between the A-B and G-H loops, which are considered to be relevant for the biological function of nitrophorins. Fast and ultrafast laser triggered ligand rebinding experiments demonstrate the pH-dependent ligand migration within the cavities and the exit route. Finally, the topological distribution of pockets located around the heme as well as from inner cavities present at the rear of the protein provides a distinctive feature in NP7, so that while a loop gated exit mechanism to the solvent has been proposed for most nitrophorins, a more complex mechanism that involves several interconnected gas hosting cavities is proposed for NP7.

No MeSH data available.


Related in: MedlinePlus

Spectral analysis.(a) Time resolved differential absorption spectra for NP7–CO (pH 7.5,T = 20°C) following femtosecond photoexcitation at 532 nm at 4 ps (black line), 200 ps (red line), 400 ps (green line) and 800 ps (blue line) delay times. (b) First spectral component (U1) obtained from the SVD analysis of the time resolved differential absorption spectra multiplied by the corresponding singular value (S1 = 0.23). (c) Comparison between the time course of the amplitudeV1 (open circles) of the main spectral component obtained from SVD, and the normalized transient absorbance at 436 nm (solid line) as a function of the delay time. The red filled circles report the amplitudeV1 of the main spectral component obtained from the experiment conducted at pH 5.5.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4482215&req=5

f8: Spectral analysis.(a) Time resolved differential absorption spectra for NP7–CO (pH 7.5,T = 20°C) following femtosecond photoexcitation at 532 nm at 4 ps (black line), 200 ps (red line), 400 ps (green line) and 800 ps (blue line) delay times. (b) First spectral component (U1) obtained from the SVD analysis of the time resolved differential absorption spectra multiplied by the corresponding singular value (S1 = 0.23). (c) Comparison between the time course of the amplitudeV1 (open circles) of the main spectral component obtained from SVD, and the normalized transient absorbance at 436 nm (solid line) as a function of the delay time. The red filled circles report the amplitudeV1 of the main spectral component obtained from the experiment conducted at pH 5.5.

Mentions: Figure 8a shows time resolved differential absorption spectra measured after photolysis of the CO adduct of NP7 at selected time delays. Transient spectra correspond to the difference between the ground state absorption spectra of NP7[FeII–CO] and NP7[FeII], with clean isosbestic points at 402 nm and 426 nm. Accordingly, only one significant spectral component is retrieved from the SVD analysis of the spectra collected between 4 ps and 1 ns (Figure 8b). As a consequence, the time course of the corresponding amplitudeV1 perfectly matches the kinetics measured at 436 nm, but with significant noise reduction, as shown inFigure 8c. Similar results are obtained when the experiment is conducted at pH 5.5, where the time resolved differential absorption spectra have the same shape as those measured at pH 7.5. Also at this pH, only one spectral component is obtained from SVD and the time course of the amplitude is shown as red solid circles inFigure 8c. It is easily observed that the time course ofV1 at acidic pH occurs with a higher rate, leading to a larger fraction of rebinding at the subnanosecond time scale.


Structure and dynamics of the membrane attaching nitric oxide transporter nitrophorin 7.

Knipp M, Ogata H, Soavi G, Cerullo G, Allegri A, Abbruzzetti S, Bruno S, Viappiani C, Bidon-Chanal A, Luque FJ - F1000Res (2015)

Spectral analysis.(a) Time resolved differential absorption spectra for NP7–CO (pH 7.5,T = 20°C) following femtosecond photoexcitation at 532 nm at 4 ps (black line), 200 ps (red line), 400 ps (green line) and 800 ps (blue line) delay times. (b) First spectral component (U1) obtained from the SVD analysis of the time resolved differential absorption spectra multiplied by the corresponding singular value (S1 = 0.23). (c) Comparison between the time course of the amplitudeV1 (open circles) of the main spectral component obtained from SVD, and the normalized transient absorbance at 436 nm (solid line) as a function of the delay time. The red filled circles report the amplitudeV1 of the main spectral component obtained from the experiment conducted at pH 5.5.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4482215&req=5

f8: Spectral analysis.(a) Time resolved differential absorption spectra for NP7–CO (pH 7.5,T = 20°C) following femtosecond photoexcitation at 532 nm at 4 ps (black line), 200 ps (red line), 400 ps (green line) and 800 ps (blue line) delay times. (b) First spectral component (U1) obtained from the SVD analysis of the time resolved differential absorption spectra multiplied by the corresponding singular value (S1 = 0.23). (c) Comparison between the time course of the amplitudeV1 (open circles) of the main spectral component obtained from SVD, and the normalized transient absorbance at 436 nm (solid line) as a function of the delay time. The red filled circles report the amplitudeV1 of the main spectral component obtained from the experiment conducted at pH 5.5.
Mentions: Figure 8a shows time resolved differential absorption spectra measured after photolysis of the CO adduct of NP7 at selected time delays. Transient spectra correspond to the difference between the ground state absorption spectra of NP7[FeII–CO] and NP7[FeII], with clean isosbestic points at 402 nm and 426 nm. Accordingly, only one significant spectral component is retrieved from the SVD analysis of the spectra collected between 4 ps and 1 ns (Figure 8b). As a consequence, the time course of the corresponding amplitudeV1 perfectly matches the kinetics measured at 436 nm, but with significant noise reduction, as shown inFigure 8c. Similar results are obtained when the experiment is conducted at pH 5.5, where the time resolved differential absorption spectra have the same shape as those measured at pH 7.5. Also at this pH, only one spectral component is obtained from SVD and the time course of the amplitude is shown as red solid circles inFigure 8c. It is easily observed that the time course ofV1 at acidic pH occurs with a higher rate, leading to a larger fraction of rebinding at the subnanosecond time scale.

Bottom Line: However, a chain-like arrangement in the crystal lattice due to a number of head-to-tail electrostatic stabilizing interactions is found in NP7.Fast and ultrafast laser triggered ligand rebinding experiments demonstrate the pH-dependent ligand migration within the cavities and the exit route.Finally, the topological distribution of pockets located around the heme as well as from inner cavities present at the rear of the protein provides a distinctive feature in NP7, so that while a loop gated exit mechanism to the solvent has been proposed for most nitrophorins, a more complex mechanism that involves several interconnected gas hosting cavities is proposed for NP7.

View Article: PubMed Central - PubMed

Affiliation: Max-Planck-Institut für Chemische Energiekonversion, Mülheim an der Ruhr, 45470, Germany.

ABSTRACT
Nitrophorins represent a unique class of heme proteins that are able to perform the delicate transportation and release of the free-radical gaseous messenger nitric oxide (NO) in a pH-triggered manner. Besides its ability to bind to phospholipid membranes, the N-terminus contains an additional Leu-Pro-Gly stretch, which is a unique sequence trait, and the heme cavity is significantly altered with respect to other nitrophorins. These distinctive features encouraged us to solve the X-ray crystallographic structures of NP7 at low and high pH and bound with different heme ligands (nitric oxide, histamine, imidazole). The overall fold of the lipocalin motif is well preserved in the different X-ray structures and resembles the fold of other nitrophorins. However, a chain-like arrangement in the crystal lattice due to a number of head-to-tail electrostatic stabilizing interactions is found in NP7. Furthermore, the X-ray structures also reveal ligand-dependent changes in the orientation of the heme, as well as in specific interactions between the A-B and G-H loops, which are considered to be relevant for the biological function of nitrophorins. Fast and ultrafast laser triggered ligand rebinding experiments demonstrate the pH-dependent ligand migration within the cavities and the exit route. Finally, the topological distribution of pockets located around the heme as well as from inner cavities present at the rear of the protein provides a distinctive feature in NP7, so that while a loop gated exit mechanism to the solvent has been proposed for most nitrophorins, a more complex mechanism that involves several interconnected gas hosting cavities is proposed for NP7.

No MeSH data available.


Related in: MedlinePlus